The present invention relates to the field of somatic embryo production, particularly to methods for the regeneration of Jatropha through somatic embryogenesis. More specifically, the present invention relates to a method and media compositions for regeneration of plants of Jatropha curcas. The method is well suited for Jatropha curcas transformation and for producing clonal planting stock useful for large scale Jatropha curcas plantation.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
The world is facing dwindling supply is fossil fuel and worsening Green House Effect. There is an urgent demand to increase production and consumption of renewable energy. Biofuels have been recognized as a national priority for many countries in their search for alternative sources to meet their energy security needs and at the same time help reduce CO2 emissions that cause the Green House Effect. The demand for biofuel has put increasing pressure on food production. For example, to satisfy the biofuel need for Germany in 2017 as mandated by the German government the entire farm land of this country would have to be used for growing bioenergy crops with no land left for food production. To ease this competition for land and to satisfy our need for renewable fuels, there is a strong need to utilize marginal land for bio-energy production.
Jatropha curcas is a small woody plant belonging to the Euphorbiaceae family. Several unique characters of Jatropha curcas make it an ideal plant for biodiesel production. These include the ability to grow on marginal land; low requirement for water; a non-food crop status; fast oil production in 1-2 years after planting compared to more than 3 years for oil palm. Accordingly, the Indonesian government has announced that they will dedicate about 3 million hectares of land for Jatropha planting in the next 5 years.
Amongst the various countries, India is the most advanced in terms of establishment of Jatropha plantations. However, the seed yield of an Indian Jatropha plantation remains low, ranging from 0.4 to 12 MT/Ha (compared with about 19Mt/Ha for palm). This difference is at least in part attributed to the lack of research in breeding and farm management in Jatropha curcas. 
The intense interest in oil from Jatropha curcas has generated enormous pressure to supply enough seeds that are homogenous and productive enough for plantation. Therefore, there is an urgent need to mass propagate elite trees. Equally urgent are methods to improve various agronomical traits of Jatropha curcas. Genetic engineering is recognized as a fast method for crop improvement. Plant transformation is essentially a two step process, i.e., delivery of genes into a host cell followed by regeneration of the transformed cell into a plant. Somatic embryogenic calli or somatic embryogenic suspension cultures is generally regarded as the most efficient method of regeneration as most of the transformed cells have already acquired the embryogenic potential that will drive them to develop into a somatic embryo quite spontaneously, a process similar to a fertilized egg cell in a zygotic embryo (Dodeman, et al., 1997).
Somatic embryos are suitable for transformation via Agrobacterium tuniefaciens (Mathews et al., 1992), microinjection (Neuhaus et al., 1987) and particle bombardment (Wilde et al., 1992). In addition, somatic embryos or somatic embryogenic calli can be cryopreserved using liquid nitrogen without loss of viability. This they are ideal materials for maintenance of germplasm as well as cell embryogenecity.
Somatic embryos are clonal in origin and thus multiplication using somatic embryos can have the potential for exceedingly high rates of vegetative increase and is therefore of considerable commercial interest. Regeneration via somatic embryogenesis is an attractive option for plant tissue culture. Somatic embryos reportedly provide more stable regenerants than shoots. Another advantage of regeneration systems using somatic embryos is their apparent single cell origin. This means that it is unlikely that regenerants are of chimerical origin, since, if a regenerant originates from a cluster of cells rather than a single sell, the plant tissues may be chimerical or unstable and produce off-types. Somatic embryogenesis has also been used successfully to mass propagation a number of plant species, e.g., banana and pines (Cote et al., 2000; Merkle and Dean, 2000). Somatic embryos may be made into synthetic seeds which reduce transportation cost and competition of seeds for oil (Conrad™, 1996).
To date, a large number of protocols for somatic embryogenesis have been developed. Some examples include the following: Eudes et al. (2006) (Pooidaea); Kasha and Simion (2001, 2004) (cereal plant); Xie and Hong (2004) (Acacia mangium); Guiltinan et al. (2001) (cacao); Trolinder et al. (1999) (cotton); Rutter et al. (1998a, 1998b) (coniferous plants); Handley and Levis (1998) (coniferous plants); Chee (1991, 1997) (squash); Becwar et al. (1995, 1996) (coniferous plants); Genovesi and Yingling (1995) (maize); Collins et al. (1991) (Glycine species); Cooley and Wilcox (1987) (sunflower); Schoofs et al. (1998) (banana); Jouenne et al. (1995) (grape); Garay et al. (2003) (agave tequilana weber); Sondahl et al. (1993) (Cacao); Armstrong and Deboer (2000) (cotton); Tuli and Mithilesh (2005) (cotton); Seabrook and Douglas (1999) (potato); Buffard-Morel et al. (1994) (coconut palm) and Cai and Ji (2005) (cotton). As illustrated in this art, various explants can be used. However, there is no culture media, culture conditions and regeneration procedures that are universally applicable. For example, Fki, et al. (2003) describes a protocol for date palm (Phoenix dactylifera) using immature florescences in which somatic embryogenesis (friable calli) was initiated on MS media supplemented with high concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) (10 mg/l), 30 mg/l adenine, 100 mg/l glutamine, 2 mg/l glycine, 30 mg/l Fe-EDTA, 100 mg/l KH2PO4, 100 mg/l myo-inositol and further embryos progression was achieved by reducing 2,4-D to 1 mg/l in either solid or liquid medium. In contrast, Cai and Ji (2005) disclose initiation of somatic embryogenesis of cotton calli, which is induced from root explants in a much simpler medium with very low concentration of 2,4-D and kinetin (0.05 mg/1 2,4-D and 0.1 mg/l kinetin), by exposing them in hormone-free, high nitrate medium after the calli initiation. In addition, dramatic changes may be found even between cultivars of the same species.
Research on somatic embryogenesis of Jatropha curcas has been very limited. Recently, preliminary results on somatic embryogenesis of Jatropha curcas using leaf tissues was reported (Ma et al., 2007). Although the author was successful in inducing somatic embryogenesis using a combination of kinetin and indolebutyric acid (IBA), less than 2% of the somatic embryos were able to convert into viable plants. Furthermore, the complete sexual life cycle using the method remains to be demonstrated. Notably, we have not been able to induce somatic embryogenesis in Jatropha curcas using the same protocol and explants despite trying three germplasm collections, including one from India.
Thus, there is a need for methods of somatic embryogensis and preparation of embryogenic liquid suspension cultures from which high efficiency plant regeneration and production of sexually fertile Jatropha curcas plants can be achieved.